Quantitative Live Imaging of Zebrafish Scale Regeneration: From Adult Fish to Signaling Patterns and Tissue Flows.

In regeneration, a damaged body part grows back to its original form. Understanding the mechanisms and physical principles underlying this process has been limited by the difficulties of visualizing cell signals and behaviors in regeneration. Zebrafish scales are emerging as a model system to investigate morphogenesis during vertebrate regeneration using quantitative live imaging. Scales are millimeter-sized dermal bone disks forming a skeletal armor on the body of the fish. The scale bone is deposited by an adjacent monolayer of osteoblasts that, after scale loss, regenerates in about 2 weeks. This intriguing regenerative process is accessible to live confocal microscopy, quantifications, and mathematical modeling. Here, I describe methods to image scale regeneration live, tissue-wide and at sub-cellular resolution. Furthermore, I describe methods to process the resulting images and quantify cell, tissue, and signal dynamics.

Studying Macrophages in the Murine Steatotic Liver Using Flow Cytometry and Confocal Microscopy.

The study of macrophage functions in the context of metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction associated steatohepatitis (MASH) has been hampered by the fact that until recently all macrophages in the liver were thought to be Kupffer cells, the resident macrophages of the liver. With the advent of single-cell technologies, it is now clear that the steatotic liver harbors many distinct populations of macrophages, likely each with their own unique functions as well as subsets of monocytes and dendritic cells which can be difficult to discriminate from one another. Here, we detail the protocols we utilize to (i) induce MASLD/MASH in mice, (ii) isolate cells from the steatotic liver, and (iii) describe reliable gating strategies, which can be used to identify the different subsets of myeloid cells. Finally, we also discuss the issue of increased autofluorescence in the steatotic liver and the techniques we use to minimize this both for flow cytometry and confocal microscopy analyses.

Identification of Immune Cells in Murine Olfactory Mucosa.

Olfactory immunology is an emerging field in the context of infectious disease and neuroimmunology, yet characterization of immune cells within the murine olfactory mucosa remains sparse. This is partially due to the difficulty in distinguishing olfactory-resident immune cells from immune cells that reside within nasal turbinate bone marrow. Using techniques like intranasal antibody labeling, we have developed methods to definitively identify olfactory immune cells via flow cytometry and immunofluorescent confocal microscopy. This protocol will describe the best practices for these methods, as well as detail how intravenous antibody labeling can be used to study the blood-olfactory barrier, an important determinant of olfactory immunity. We also include validated markers for the identification of major olfactory immune populations.

Optical Activation of Photoswitchable TRPC Ligands in the Mammalian Olfactory System Using Laser Scanning Confocal Microscopy.

The transient receptor potential canonical (TRPC) ion channels play important biological roles, but their activation mechanisms are incompletely understood. Here, we describe recent methodological advances using small molecular probes designed for photopharmacology of TRPC channels by focusing on results obtained from the mouse olfactory system. These studies developed and used photoswitchable diacylglycerol (DAG) analogs for ultrarapid activation of native TRPC2 channels in vomeronasal sensory neurons and type B cells of the main olfactory epithelium. Further studies investigated the role of TRPC5 channels in prolactin regulation of dopamine neurons in the arcuate nucleus of the hypothalamus. Here, the first photoswitchable TRPC5 modulator, BTDAzo, was developed and shown to control endogenous TRPC5-based neuronal Ca2+ responses in mouse brain slices. Thus, photoswitchable reagents are rapidly gaining widespread recognition for investigating various types of TRPC channels including TRPC2, TRPC3, TRPC5, and TRPC6, enabling to gain new insights into the gating mechanisms and functions of these channels.

Open-Source Automated Segmentation of Neuronal Structures in Corneal Confocal Microscopy Images of the Subbasal Nerve Plexus With Accuracy on Par With Human Segmentation.

PURPOSE: The aim of this study was to perform automated segmentation of corneal nerves and other structures in corneal confocal microscopy (CCM) images of the subbasal nerve plexus (SNP) in eyes with ocular surface diseases (OSDs). METHODS: A deep learning-based 2-stage algorithm was designed to perform segmentation of SNP features. In the first stage, to address applanation artifacts, a generative adversarial network-enabled deep network was constructed to identify 3 neighboring corneal layers on each CCM image: epithelium, SNP, and stroma. This network was trained/validated on 470 images of each layer from 73 individuals. The segmented SNP regions were further classified in the second stage by another deep network as follows: background, nerve, neuroma, and immune cells. Twenty-one-fold cross-validation was used to assess the performance of the overall algorithm on a separate data set of 207 manually segmented SNP images from 43 patients with OSD. RESULTS: For the background, nerve, neuroma, and immune cell classes, the Dice similarity coefficients of the proposed automatic method were 0.992, 0.814, 0.748, and 0.736, respectively. The performance metrics for automatic segmentations were statistically better or equal as compared to human segmentation. In addition, the resulting clinical metrics had good to excellent intraclass correlation coefficients between automatic and human segmentations. CONCLUSIONS: The proposed automatic method can reliably segment potential CCM biomarkers of OSD onset and progression with accuracy on par with human gradings in real clinical data, which frequently exhibited image acquisition artifacts. To facilitate future studies on OSD, we made our data set and algorithms freely available online as an open-source software package.

Clinical and in vivo confocal microscopy characteristics of Candida keratitis following keratoplasty.

BACKGROUND: We present six patients who developed Candida keratitis postoperatively. The clinical features, diagnostic testing including in vivo confocal microscopy, and outcomes are presented. METHODS: Six patients who developed Candida keratitis following penetrating and endothelial keratoplasty, were referred to Tianjin Medical University Eye Hospital between 2018 to 2021.The diagnosis was established following cultures of either corneal scraping or biopsy. In vivo confocal microscopy examination was also performed to confirm the diagnosis and characterize the morphology, distribution and the depth of Candida spp. All patients were treated with topical voriconazole (VCZ) 1% and natamycin (NTM) 5%. Patients with mid/deep stromal keratitis or interface infection were treated additionally with intrastromal or interface VCZ irrigation (0.05 mg/0.1mL). RESULTS: The cultures of corneal scrapings (4 cases) or biopsies (2 cases) were all positive for Candida spp. In vivo confocal microscopy examination was positive for fungal elements in five of the six patients. The infection resolved in five of the six patients. The patients' final uncorrected visual acuity (UCVA) ranged from hand movements (HM) to 20/80. CONCLUSION: In vivo confocal microscopy is a useful non-invasive clinical technique for confirming the diagnosis of Candida keratitis. Intrastromal and interface irrigated VCZ injections are effective treatment options.

Ferrocene/ ß-cyclodextrin based supramolecular nanogels as theranostic systems.

A supramolecular redox responsive nanogel (NG) with the ability to sense cancer cells and loaded with a releasing therapeutic agent was synthesized using hostguest interactions between polyethylene glycol-grafted-ß-cyclodextrin and ferrocene boronic acid. Cyclic voltammetry matched with other spectroscopy and microscopy methods provided strong indications regarding host-guest interactions and formation of the NG. Moreover, the biological properties of the NG were evaluated using fluorescence silencing, confocal laser scanning microscopy, and cell toxicity assays. Nanogel with spherical core-shell architecture and 100-200 nm sized nanoparticles showed high encapsulation efficiency for doxorubicin (DOX) and luminol (LU) as therapeutic and sensing agents. High therapeutic and sensing efficiencies were manifested by complete release of DOX and dramatic quenching of LU fluorescence triggered by 0.05 mM H2O2 (as an ROS component). The NGs showed high ROS sensitivity. Taking advantage of a high loading capacity, redox sensitivity, and biocompatibility, the NGs can be used as strong theranostic systems in inflammation-associated diseases.

Effects of thickness and polishing treatment on the translucency and opalescence of six dental CAD-CAM monolithic restorative materials: an in vitro study.

BACKGROUND: Computer-aided design and computer-aided manufacturing (CAD-CAM) materials for prosthetic is gaining popularity in dentistry. However, limited information exists regarding the impact of thickness and roughening treatment on the optical properties of contemporary CAD-CAM restorative materials. This study aimed to quantitatively evaluate the translucency and opalescence of six dental CAD-CAM materials in response to different thicknesses and roughening treatments. METHODS: Six dental CAD-CAM materials, lithium disilicate glass-ceramic (IPS e.max CAD, LS), polymer-infiltrated ceramic (VITA Enamic, VE), resin-nano ceramic glass-ceramic (LAVA Ultimate, LU), polymethyl methacrylate (Telio CAD, TE), and two zirconia reinforced lithium silicate (VITA Suprinity, VS, and Celtra Duo, CD), in shade A2 were prepared as 12 × 12mm2 specimens of four thicknesses (0.5mm, 1.0mm, 1.5mm, and 2.0mm) (N = 240, n = 10). After three different treatments (polished, roughened by SiC P800-grit, and SiC P300-grit), the translucency parameter (TP00) and opalescence parameter (OP) were measured with a spectrophotometer (VITA Easyshade V). The surface roughness was analyzed with a shape measurement laser microscope. The data were analyzed using a MANOVA, post hoc Tukey-Kramer test, the t test, and regression analysis (α = .05). RESULTS: The TP00 and OP were significantly influenced by material type, thickness and roughening treatment (P < .05). TP00 showed a continues decline with increasing thicknesses, while the variations of OP were material-dependent. TP00 ranged from 37.80 (LS in 0.5mm) to 5.66 (VS in 2.0mm), and OP ranged from 5.66 (LU in 0.5mm) to 9.55 (VS in 0.5mm). The variations in TP00 of all materials between adjacent thicknesses ranged from 2.10 to 15.29, exceeding the acceptable translucency threshold except for LU. Quadratic and logarithmic regression curves exhibited the best fit for TP00 among the materials. Compared to polished specimens, rougher specimens exhibited lower TP00 and higher OP in all materials except for LS (P < 0.05). Roughening with P300-grit decreased TP00 and OP by an average of 2.59 and 0.43 for 0.5mm specimens, and 1.26 and 0.25 for 2.0mm specimens, respectively. CONCLUSIONS: Variations in translucency caused by thickness and roughening treatment were perceptible and may be clinically unacceptable. Careful consideration should be given to the selection of CAD-CAM materials based on their distinct optical properties.

Evaluating models and assessment techniques for understanding oral biofilm complexity.

Oral biofilms are three-dimensional (3D) complex entities initiating dental diseases and have been evaluated extensively in the scientific literature using several biofilm models and assessment techniques. The list of biofilm models and assessment techniques may overwhelm a novice biofilm researcher. This narrative review aims to summarize the existing literature on biofilm models and assessment techniques, providing additional information on selecting an appropriate model and corresponding assessment techniques, which may be useful as a guide to the beginner biofilm investigator and as a refresher to experienced researchers. The review addresses previously established 2D models, outlining their advantages and limitations based on the growth environment, availability of nutrients, and the number of bacterial species, while also exploring novel 3D biofilm models. The growth of biofilms on clinically relevant 3D models, particularly melt electrowritten fibrous scaffolds, is discussed with a specific focus that has not been previously reported. Relevant studies on validated oral microcosm models that have recently gaining prominence are summarized. The review analyses the advantages and limitations of biofilm assessment methods, including colony forming unit culture, crystal violet, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt assays, confocal microscopy, fluorescence in situ hybridization, scanning electron microscopy, quantitative polymerase chain reaction, and next-generation sequencing. The use of more complex models with advanced assessment methodologies, subject to the availability of equipment/facilities, may help in developing clinically relevant biofilms and answering appropriate research questions.

[Research progress on imagine diagnosis of limbal stem cell deficiency].

Limbal stem cell deficiency (LSCD) is an ocular surface disease resulting from a reduction and/or dysfunction of limbal stem cells. The symptoms of LSCD are non-specific and can be difficult to distinguish from other ocular surface diseases through slit-lamp examination. Impression cytology is currently considered the gold standard for LSCD diagnosis; however,it is a qualitative method with low sensitivity. Nonetheless,emerging imaging techniques offer quantitative diagnosis and staging of LSCD. This review article examines four imaging methods and their associated parameters for diagnosing LSCD: optical coherence tomography,which measures corneal epithelial thickness; optical coherence tomography angiography,which detects corneal neovascularization; in vivo confocal microscopy,which measures corneal epithelial thickness,subbasal nerve density,and corneal basal cell density; and future applications of full-field/spectral-domain optical coherence tomography.

Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence.

Inflammasomes are crucial sentinels of the innate immune system that sense clues of infection, cellular stress, or metabolic imbalances. Upon activation, the inflammasome sensor (e.g., NLRP3) recruits the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). ASC rapidly oligomerizes to form a micron-sized structure termed "ASC speck." These are crucial for the activation of caspase-1 and downstream inflammatory signals released following a specific form of lytic cell death called pyroptosis. Hence, due to their considerably large size, ASC specks can be easily visualized by microscopy as a simple upstream readout for inflammasome activation. Here, we provide three detailed protocols for imaging ASC specks: (1) live-cell imaging of macrophage cell lines expressing a fluorescent protein fusion form of ASC, (2) imaging of human primary cells using immunofluorescence staining of endogenous ASC, and (3) visualization and quantification of specks on a single-cell level using imaging flow cytometry.

The unplumatellid Plumatella fruticosa found its home: Hirosella gen. nov. morphological arguments for the systematic placement of a freshwater bryozoan.

Bryozoans are colonial, suspension-feeding lophotrochozoans. The phylum consists of the large group of chiefly marine Myolaemata and the exclusively limnic Phylactolaemata. Each colony consists of individual zooids that comprise the protective cystid and the retractable polypide. Phylactolaemates are a small group of approximately 90 species in 6 families. They feature a body wall, that can either be gelatinous, as in the families Stephanellidae, Lophopodidae, Cristatellidae and Pectinatellidae, or encrusted, as in Plumatellidae and Fredericellidae. Morphological investigations of the most specious plumatellids are rare and focus on few species. Plumatella fruticosa is of particular interest in this regard, as it shows a mosaic of plumatellid and fredericellids characters. The most recent phylogeny clusters P. fruticosa with cristatellids and pectinatellids as sister groups to fredericellids. Hence, there is considerable doubt, whether P. fruticosa is truly a plumatellid. Therefore, this study aims to reinvestigate the morphology of P. fruticosa with confocal microscopy and section-based three-dimensional reconstruction. The new data show that P. fruticosa has numerous conspicuous stumps from fragmented proliferation buds, which are otherwise only known from fredericellids. Like fredericellids, P. fruticosa grows erect, but in contrast, has a horseshoe-shaped lophophore and floatoblasts. Besides the proportions of the lophophore, the tentacle sheath and digestive tract resemble a fredericellid-like situation. Myoanatomical details like the pronounced longitudinal muscles of the vestibular wall and tentacle sheath differ from plumatellids and favour the recently proposed scenario, which places P. fruticosa next to Pectinatellidae and Cristatellidae. In addition, the intertentacular membrane of P. fruticosa shows structural similarity to cristatellids as it is attached to the tentacles via lamellae. Taking all aspects into account, we erect a new family: Hirosellidae fam. nov. including the new genus Hirosella gen. nov.

The liquid-to-solid transition of FUS is promoted by the condensate surface.

A wide range of macromolecules can undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed reversibly, and carry out essential functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics, and mechanics in time and space, that single-component FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases coexist simultaneously within the same condensate, resulting in highly inhomogeneous structures. Furthermore, our results show that this transition originates at the interface between the condensate and the dilute continuous phase, and once initiated, the gelation process propagates toward the center of the condensate. To probe such spatially inhomogeneous rheology during condensate aging, we use a combination of established micropipette aspiration experiments together with two optical techniques, spatial dynamic mapping and reflective confocal dynamic speckle microscopy. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a critical element in driving pathological protein aggregation.

High-speed multiplane confocal microscopy for voltage imaging in densely labeled neuronal populations.

Genetically encoded voltage indicators (GEVIs) hold immense potential for monitoring neuronal population activity. To date, best-in-class GEVIs rely on one-photon excitation. However, GEVI imaging of dense neuronal populations remains difficult because out-of-focus background fluorescence produces low contrast and excess noise when paired with conventional one-photon widefield imaging methods. To address this challenge, we developed an imaging system capable of efficient, high-contrast GEVI imaging at near-kHz rates and demonstrate it for in vivo and ex vivo imaging applications in the mouse neocortex. Our approach uses simultaneous multiplane imaging to monitor activity within contiguous tissue volumes with no penalty in speed or requirement for high excitation power. This approach, multi-Z imaging with confocal detection (MuZIC), permits high signal-to-noise ratio voltage imaging in densely labeled neuronal populations and is compatible with imaging through micro-optics. Moreover, it minimizes artifacts associated with concurrent imaging and optogenetic photostimulation for all-optical electrophysiology.

Reliability of confocal corneal microscopy for measurement of dendritic cell density in suspected small fiber neuropathy.

INTRODUCTION/AIMS: Dendritic cells (DCs) and their contacts with corneal nerves are described in animal models of nerve damage. Dendritic cell density (DCD) is a potential marker of immune activity in suspected small-fiber neuropathy (SFN). Here, we aim to evaluate the intra- and inter-rater reliability of DCD measurements in suspected SFN. METHODS: This retrospective study collected DCD from confocal microscopy images from the corneal sub-basal epithelium of the eye from 48 patients (mean age 49.6 ± 12.1 y, 61% female). Two examiners, each blinded to the other's examinations and measurements, assessed DCD to evaluate inter-rater reliability. For intra-rater reliability, the first examiner performed a second measurement after 14 days. DCs were classified into two cell morphological subtypes: mature and immature. RESULTS: Test-retest reliability for total DCD showed excellent agreement, with an intraclass correlation coefficient of 0.96 and inter-rater reliability intraclass correlation coefficient of 0.77. The immature cell subtype showed excellent intra-rater reliability but lower inter-rater reliability. DISCUSSION: We found that DCD measurements in the corneal sub-basal epithelium are sufficiently reliable for consideration in clinical studies of patients with suspected SFN.

Needle-based confocal laser endomicroscopy for real-time granuloma detection.

BACKGROUND AND OBJECTIVE: Needle-based confocal laser endomicroscopy (nCLE) allows real-time microscopic imaging at the needle tip. nCLE malignancy criteria are used for tool-in-lesion confirmation during bronchoscopic lung nodule analysis. However, to date, nCLE criteria for granulomas are lacking. The aim was to identify and validate nCLE granuloma criteria and assess if blinded raters can distinguish malignant from granulomatous nCLE videos. METHODS: In patients with suspected sarcoidosis, nCLE-imaging of mediastinal lymph nodes was performed during endoscopic ultrasound procedures, followed by needle aspiration. nCLE granuloma criteria were identified by comparison with pathology and final diagnoses. Additionally, nCLE-videos of granulomatous lung nodules part of prospective trials and clinical care were compared to the proposed nCLE granuloma criteria. Blinded raters validated nCLE videos of sarcoid and reactive mediastinal lymph nodes and malignant and granulomatous lung nodules twice. RESULTS: Granuloma criteria were identified (brighter-toned, homogeneous and well-demarcated lesions) based on nCLE-imaging in 14 sarcoidosis patients. Raters evaluated 26 nCLE-videos obtained in lymph nodes (n = 15 sarcoidosis; n = 11 reactive and total of 260 ratings). Granuloma criteria were recognized with 88% accuracy. The inter-observer (κ = 0.63, 95% CI 0.54-0.72) and intra-observer reliability (κ = 0.70 ± 0.06) were substantial. Based on 12 nCLE-videos obtained in lung nodules (n = 4 granulomas, n = 6 malignancy, n = 2 malignancy + granulomas and total of 120 ratings) granuloma and malignancy criteria were recognized with 92% and 75% accuracy. CONCLUSION: nCLE imaging facilitates real-time granuloma visualization. Blinded raters accurately and consistently recognized granulomas on nCLE-imaging and distinguished nCLE granuloma criteria from malignancy. Our data show the potential of nCLE as a real-time bronchoscopic guidance tool for lung nodule analysis.

Effect of Imposing Spatial Constraints on Low Molecular Weight Gels.

We outline the effect of imposing spatial constraints during gelation on hydrogels formed by dipeptide-based low molecular weight gelators. The gels were formed via either a solvent switch or a change in pH and formed in different sized vessels to produce gels of different thickness while maintaining the same volume. The different methods of gelation led to gels with different underlying microstructure. Confocal microscopy was used to visualize the resulting microstructures, while the corresponding mechanical properties were probed via cavitation rheology. We show that solvent-switch-triggered gels are sensitive to imposed spatial constraints, in both altered microstructure and mechanical properties, while their pH-triggered equivalents are not. These results are significant because it is often necessary to form gels of different thicknesses for different analytical techniques. Also, gels of different thicknesses are utilized between various applications of these materials. Our data show that it is important to consider the spatial constraints imposed in these situations.